U.S. patent number 10,987,425 [Application Number 15/025,127] was granted by the patent office on 2021-04-27 for liquid formulation of long-acting human growth hormone immunoglobulin conjugate.
This patent grant is currently assigned to HANMI PHARM. CO., LTD.. The grantee listed for this patent is HANMI PHARM. CO., LTD.. Invention is credited to Sung Min Bae, Ho Taek Im, Hyun Uk Kim, Sang Yun Kim, Se Chang Kwon, Hyung Kyu Lim, Hwa Peoung Rim.
![](/patent/grant/10987425/US10987425-20210427-D00001.png)
![](/patent/grant/10987425/US10987425-20210427-D00002.png)
![](/patent/grant/10987425/US10987425-20210427-D00003.png)
United States Patent |
10,987,425 |
Rim , et al. |
April 27, 2021 |
Liquid formulation of long-acting human growth hormone
immunoglobulin conjugate
Abstract
The present invention relates to a sustained type human growth
hormone conjugate preparation comprising: a sustained type human
growth hormone (hGH) conjugate resulting from conjugation between
the immunoglobulin Fc region and a human growth hormone (hGH)
constituting a bioactive peptide; a buffer solution; a nonionic
surfactant; and a sugar alcohol. More specifically, the present
invention relates to a sustained type human growth hormone
conjugate freeze dried preparation and liquid preparation, to a
production method for the freeze dried preparation, to a method of
reconstituting the freeze dried preparation, and to a kit
comprising the freeze dried preparation and a reconstituting
solution.
Inventors: |
Rim; Hwa Peoung (Seoul,
KR), Kim; Hyun Uk (Busan, KR), Im; Ho
Taek (Yongin-si, KR), Kim; Sang Yun (Gimpo-si,
KR), Lim; Hyung Kyu (Hwaseong-si, KR), Bae;
Sung Min (Seongnam-si, KR), Kwon; Se Chang
(Seoul, KR) |
Applicant: |
Name |
City |
State |
Country |
Type |
HANMI PHARM. CO., LTD. |
Hwaseong-si |
N/A |
KR |
|
|
Assignee: |
HANMI PHARM. CO., LTD.
(Hwaseong-si, KR)
|
Family
ID: |
1000005513058 |
Appl.
No.: |
15/025,127 |
Filed: |
September 26, 2014 |
PCT
Filed: |
September 26, 2014 |
PCT No.: |
PCT/KR2014/009059 |
371(c)(1),(2),(4) Date: |
March 25, 2016 |
PCT
Pub. No.: |
WO2015/046974 |
PCT
Pub. Date: |
April 02, 2015 |
Prior Publication Data
|
|
|
|
Document
Identifier |
Publication Date |
|
US 20160213789 A1 |
Jul 28, 2016 |
|
Foreign Application Priority Data
|
|
|
|
|
Sep 27, 2013 [KR] |
|
|
10-2013-0115177 |
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K
9/19 (20130101); A61K 9/0019 (20130101); A61K
38/27 (20130101); A61K 47/183 (20130101); A61K
47/26 (20130101); A61K 47/6883 (20170801); A61K
47/12 (20130101); A61K 47/6811 (20170801); A61K
47/68 (20170801) |
Current International
Class: |
A61K
38/27 (20060101); A61K 9/19 (20060101); A61K
47/12 (20060101); A61K 47/18 (20170101); A61K
47/26 (20060101); A61K 47/68 (20170101); A61K
9/00 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
|
|
|
|
|
|
|
103068366 |
|
Apr 2013 |
|
CN |
|
2 606 908 |
|
Jun 2013 |
|
EP |
|
H10-265404 |
|
Oct 1998 |
|
JP |
|
2001-524360 |
|
Dec 2001 |
|
JP |
|
2005-232177 |
|
Sep 2005 |
|
JP |
|
10-2000-0010630 |
|
Feb 2000 |
|
KR |
|
10-0273053 |
|
Dec 2000 |
|
KR |
|
10-0567902 |
|
Apr 2006 |
|
KR |
|
10-2006-0106486 |
|
Oct 2006 |
|
KR |
|
10-0725315 |
|
Jun 2007 |
|
KR |
|
10-2012-007182 |
|
Jan 2012 |
|
KR |
|
2190129 |
|
Sep 2002 |
|
RU |
|
2229288 |
|
May 2004 |
|
RU |
|
2011/073234 |
|
Jun 2011 |
|
WO |
|
2012/008779 |
|
Jan 2012 |
|
WO |
|
2013/147559 |
|
Oct 2013 |
|
WO |
|
Other References
International Searching Authority, International Search Report for
PCT/KR2014/009059 dated Jan. 19, 2015 [PCT/ISA/210]. cited by
applicant .
International Searching Authority, Written Opinion for
PCT/KR2014/009059 dated Jan. 19, 2015 [PCT/ISA/237]. cited by
applicant .
European Patent Office; Communication dated May 2, 2017 in
counterpart European application No. 14847086.7. cited by applicant
.
State Intellectual Property Office of the P.R.C.; Communication
dated Apr. 23, 2018 in counterpart application No. 201480064028.X.
cited by applicant .
Taiwanese Patent Office; Communication dated Apr. 11, 2018 in
counterpart application No. 103133464. cited by applicant .
European Patent Office; Communication dated Mar. 8, 2018 in
counterpart application No. 14847086.7. cited by applicant .
Carpenter, J. F., et al., "Rational Design of Stable Lyophilized
Protein Formulations: Theory and Practice", Pharmaceutical
Biotechno., vol. 13, Jan. 1, 2002, 23 pages, XP009053337. cited by
applicant .
Japanese Patent Offcie; Communication dated Jul. 2, 2018 in
counterpart application No. 2016-518140. cited by
applicant.
|
Primary Examiner: Saoud; Christine J
Attorney, Agent or Firm: Sughrue Mion, PLLC
Claims
The invention claimed is:
1. A liquid formulation of a long-acting human growth hormone (hGH)
conjugate, wherein the liquid formulation comprises a
pharmaceutically effective amount of a long-acting human growth
hormone conjugate in which the physiologically active human growth
hormone is linked to an immunoglobulin Fc region and an
albumin-free stabilizer, wherein the liquid formulation comprises a
buffer, a non-ionic surfactant, a sugar alcohol, and preservative,
wherein the sugar alcohol is included with a concentration ranging
from 2% (w/v) to 4.5% (w/v); wherein the preservative is benzyl
alcohol, m-cresol, or phenol; wherein the formulation does not
comprise sodium chloride; wherein the buffer is a citrate buffer,
an acetate buffer, or a histidine buffer; wherein the non-ionic
surfactant is polysorbate 80; wherein the sugar alcohol is mannitol
or sorbitol; and wherein the concentration of the long-acting hGH
conjugate ranges from 58.5 to 60 mg/mL.
2. The liquid formulation of a long-acting human growth hormone
conjugate according to claim 1, wherein the pH of the buffer ranges
from 5.0 to 6.0.
3. The liquid formulation of a long-acting human growth hormone
conjugate according to claim 1, wherein the concentration of the
non-ionic surfactant ranges from 0.001% (w/v) to 0.05% (w/v) of the
total volume of the formulation.
4. The liquid formulation of a long-acting human growth hormone
conjugate according to claim 1, wherein the conjugate is in such a
form that the human growth hormone is linked to the immunoglobulin
Fc via a non-peptidyl polymer as a linker or via genetic
recombination.
5. The liquid formulation of a long-acting human growth hormone
conjugate according to claim 4, wherein the non-peptidyl polymer is
a biodegradable polymer.
6. The liquid formulation of a long-acting human growth hormone
conjugate according to claim 5, wherein the biodegradable polymer
is selected from the group consisting of polyethylene glycol,
polypropylene glycol, a copolymer of ethylene glycol and propylene
glycol, a polyoxyethylated polyol, polyvinyl alcohol, a
polysaccharide, dextran, polyvinyl ethyl ether, polylactic acid
(PLA) and polylactic-glycolic acid (PLGA), a lipid polymer, chitin,
hyaluronic acid and a combination thereof.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
This application is a National Stage of International Application
No. PCT/KR2014/009059 filed Sep. 26, 2014, claiming priority based
on Korean Patent Application No. 10-2013-0115177 filed Sep. 27,
2013.
TECHNICAL FIELD
The present invention relates to a formulation of a long-acting
human growth hormone conjugate, comprising a long-acting human
growth hormone (hGH) conjugate in which the human growth hormone as
a physiologically active peptide is linked to an immunoglobulin Fc
region, a buffer, a non-ionic surfactant and a sugar alcohol,
specifically a lyophilized formulation and a liquid formulation of
a long-acting human growth hormone conjugate, a method for
preparing the lyophilized formulation, a method for reconstituting
the lyophilized formulation and a kit comprising the lyophilized
formulation and a solution for reconstitution.
BACKGROUND ART
Human growth hormone (hereinafter referred to as "hGH") is a
polypeptide hormone consisting of 191 amino acids having a
molecular weight of about 22,000, being secreted from the anterior
pituitary gland. The human growth hormone has been mostly used for
the treatment of pediatric pituitary dwarfism. Conventionally, hGH
extracted from the human pituitary gland has been used but only a
very limited number of people have been treated due to its limited
supply. Also, since the reports of Creutzfeldt-Jacob disease, a
degenerative neurological disorder, found in some of the patients
treated with the hGH extracted from the pituitary gland, the use of
hGH extracted from the pituitary glands has been banned. Currently,
the development of genetic engineering techniques has enabled
production of hGH in E. coli and yeast, and the biosynthetic hGH
medicines produced therefrom have been approved in several
countries since 1985 and become commercially available after
passing toxicological and clinical tests.
In general, polypeptides such as hGH have low stability and thus
are easily denatured. Also, they are readily degraded by serum
proteases and removed by the kidneys or liver. Thus, protein drugs
containing polypeptides as pharmaceutical ingredient have to be
frequently administered to patients to maintain its blood
concentration and titer. However, since the protein drugs are often
administered in the form of injection, frequent injection of the
protein drugs to maintain the optimal blood concentration of the
active polypeptides causes a lot of pain to the patients. To solve
these problems, there have been many attempts to increase the
stability of a protein drug in blood and maintain its blood
concentration at high level for a long period of time to maximize
the therapeutic effects of the medicine.
Recently, Korean Patent No. 10-0567902 (Physiologically Active
Polypeptide Conjugate Having Improved In Vivo Durability) and
Korean Patent No. 10-0725315 (Protein Complex Using An
Immunoglobulin Fragment And Method For The Preparation Thereof)
disclosed conjugates prepared by linking physiologically active
polypeptides with an immunoglobulin Fc region and a non-peptidyl
polymer, as long-acting formulations of protein drugs, enabling
both a minimal reduction of protein activity and an increase in
protein stability. According to these methods, hGH may be used as a
physiologically active polypeptide to prepare a long-acting hGH
conjugate. For commercializing the drug containing the long-acting
hGH conjugate, it is essential to prevent physicochemical changes
such as denaturation, aggregation, adsorption, or hydrolysis due to
degradation induced by light, heat or impurities in additives
during storage and transport processes, while retaining the in-vivo
activities of hGH. Since the long-acting hGH conjugate has a larger
size and increased molecular weight compared to a hGH polypeptide,
it is difficult to stabilize the conjugate.
Lyophilization (freeze-drying) is commonly used to preserve
proteins by removing water from the protein preparation of
interest. Lyophilization is a process by which the material to be
dried is first frozen and then the ice or frozen solvent is removed
by sublimation. An excipient may be included in a pre-lyophilized
formulation to maintain or enhance protein stability during the
lyophilization process or to improve stability of the lyophilized
product during storage. However, the composition of a lyophilized
formulation applicable to one protein is often not applicable to
other proteins due to the difference in properties of the proteins
to be preserved. Specifically, different proteins may be
inactivated under different conditions during the storage,
lyophilization and reconstitution processes owing to their
different chemical properties. That is, the enhancement in
stability provided by the materials used for stabilization is not
identical for different proteins and, accordingly, the suitable
ratios, concentrations and kinds of the stabilizers used to provide
stability during the storage, lyophilization and reconstitution
processes vary depending on the physicochemical properties of the
proteins. When different stabilizers are used in combination, an
unwanted negative effect may be derived due to their competition or
adverse reactions and an unexpected effect may occur due to the
change in the nature or concentration of the protein during the
lyophilization or storage processes. Therefore, protein
stabilization requires a lot of effort and precautions.
Particularly, since a long-acting hGH conjugate having improved in
vivo durability and stability has a form in which the human growth
hormone as a physiologically active peptide is linked to the
immunoglobulin Fc region, its molecular weight and volume differs
greatly from those of the human growth hormone. Therefore, a
special composition is required for stabilizing the protein. Also,
since each of the physiologically active peptide hGH and the
immunoglobulin Fc region has different physicochemical properties,
they should be stabilized simultaneously. However, as described
above, different peptides or proteins may be gradually inactivated
under different ratios and conditions due to the difference in
their physicochemical properties. Also, when stabilizers suitable
for each peptide or protein are used simultaneously, they may cause
adverse results due to competitive interactions between them and
side effects. Furthermore, as the properties and concentration of
the stored protein may change during its storage, the stabilizers
may exhibit unexpected side effects. Therefore, for a long-acting
hGH conjugate, it is difficult to find a composition suitable for a
stabilizer capable of stabilizing both the physiologically active
peptide hGH and the immunoglobulin Fc region simultaneously. In
addition, for a lyophilized formulation, the methods of
lyophilization and reconstituting should be controlled in various
ways to maintain protein stability and activity upon
reconstitution. These methods may also vary depending on the
composition of the formulation and the protein used thereof.
Additionally, when a lyophilized formulation comprises a protein at
high concentration, the protein may aggregate during lyophilization
because of the high concentration, and its handling also becomes
difficult. Therefore, a protein at high concentration had been
conventionally obtained by preparing a lyophilized formulation
comprising the protein at low concentration and then reconstituting
it with a small volume instead of performing lyophilization
followed by reconstitution of the high-concentration protein.
However, if the protein is reconstituted with a small volume, not
only the protein but also other ingredients included therein become
too concentrated and too hypertonic for the formulation to be
directly applicable to patients. Accordingly, there is a need for
the development of a formulation that allows lyophilizing of the
high-concentration protein as it is.
Recently, formulations of proteins and peptides that can be used
repeatedly for the patients' convenience have been developed.
However, these multi-use formulations should contain a preservative
to prevent microbial contamination after repeated administration
and prior to disposal. The multi-use formulation containing a
preservative has a few advantages over a single-use formulation.
For example, as for the single-use formulation, a large amount of
drug may be wasted depending on the dosage, which may be reduced
when the multi-use formulation is used. Furthermore, the multi-use
formulation can be used several times without the concern about
microbial growth during a given time period and, since it can be
supplied in a single container, packaging can be minimized, leading
to economic benefits. However, use of the preservative may affect
the protein stability. The most well-known problem associated with
the use of a preservative is formation of precipitates.
Precipitation of the protein may reduce the therapeutic effect of
the drug and induce an unexpected immune response when administered
to the body. Therefore, it is critical to select an appropriate
type and concentration of the preservative that maintain the
ability of microbial contamination without affecting the protein
stability.
In general, a formulation in solution state is developed in a
syringe form. The most commonly-used type is a prefilled syringe,
and a more convenient autoinjector is also frequently used. In
addition, a pen injector which allows automated injection of a
required dosage to a patient is used mainly for growth hormone,
insulin, etc. Although these injection devices are convenient for
administration of formulations in solution state, they cannot be
used for the drugs which must be lyophilized because of low
stability.
In general, a lyophilized formulation is prepared in a reinforced
glass vial separately from a solvent for dissolution. The two are
mixed to dissolve the lyophilized formulation immediately prior to
injection using a syringe. The recent trend is from a lyophilized
vial (e.g., a reinforced glass vial) toward a single-use or
multi-use syringe for the patients' convenience. Examples include
the dual chamber cartridge of Vetter (Germany).
DISCLOSURE
Technical Problem
Under this background, the inventors of the present invention have
made efforts to develop a lyophilized formulation capable of
maintaining the stability of a long-acting human growth hormone
conjugate during the lyophilization process and capable of storing
it for a long period of time and a liquid formulation capable of
stably storing a long-acting human growth hormone conjugate. As a
result, they have found that when a stabilizer comprising a buffer,
a sugar alcohol and a non-ionic surfactant is used, the stability
of a long-acting hGH conjugate is increased during lyophilization
and storage, and thus a cost-effective and stable liquid
formulation could be prepared. Also, it was confirmed that when the
concentration of the long-acting hGH conjugate is 10-58.5 mg/mL, a
sodium chloride-free liquid formulation with superior stability can
be provided. Furthermore, it was confirmed that the lyophilized
formulation of the present invention is not only stable during
storage and transportation but also it has appropriate osmotic
pressure and stability for subcutaneous injection when
reconstituted. In addition, it was confirmed that the lyophilized
formulation can be used as a multi-use formulation since it
maintains stability even when a preservative is included.
Technical Solution
The present invention is directed to providing a formulation of a
long-acting human growth hormone conjugate, comprising a
long-acting human growth hormone (hGH) conjugate in which the human
growth hormone (hGH) as a physiologically active peptide is linked
to an immunoglobulin Fc region, a buffer, a non-ionic surfactant
and a sugar alcohol.
The present invention is also directed to providing a lyophilized
formulation of a long-acting hGH conjugate, comprising a
lyophilized mixture of an aqueous solution comprising a long-acting
human growth hormone conjugate in which the hGH as a
physiologically active peptide is linked to an immunoglobulin Fc
region and an albumin-free solution comprising a buffer, a
non-ionic surfactant and a sugar alcohol.
The present invention is also directed to providing a liquid
formulation of a long-acting hGH conjugate, comprising a
pharmaceutically effective amount of a long-acting hGH conjugate in
which the hGH as a physiologically active peptide is linked to an
immunoglobulin Fc region and an albumin-free stabilizer, wherein
the stabilizer comprises a buffer, a non-ionic surfactant and a
sugar alcohol.
The present invention is also directed to providing a method for
preparing the formulations.
The present invention is also directed to providing a method for
reconstituting the lyophilized formulation, comprising adding a
solution for reconstitution to the lyophilized mixture of an
aqueous solution comprising a long-acting human growth hormone
conjugate in which the human growth hormone as a physiologically
active peptide is linked to an immunoglobulin Fc region and an
albumin-free solution comprising a buffer, a non-ionic surfactant
and a sugar alcohol.
The present invention is also directed to providing a kit
comprising the lyophilized formulation of a long-acting hGH
conjugate.
Advantageous Effects
Since the formulation of a long-acting hGH conjugate of the present
invention does not comprises human serum albumin or any potentially
hazardous factors, there is no concern of viral contamination. In
addition, the formulation allows for a high stability of the
long-acting hGH conjugate which is prepared by linking the hGH
polypeptide to the immunoglobulin Fc region, thus having a larger
molecular weight when compared to a wild-type and increased in vivo
durability. In particular, the lyophilized formulation provides
superior stability not only during lyophilization but also after
reconstitution, and also maintains stability even when it contains
a preservative, thus being useful as a formulation for multiple
administrations.
DESCRIPTION OF DRAWINGS
FIG. 1 shows a temperature gradient used in a lyophilization
process of the present invention.
FIG. 2 shows a temperature gradient used in a lyophilization
process of the present invention wherein primary drying is divided
into two stages.
FIG. 3 shows a Vetter's dual chamber cartridge.
BEST MODE
In an aspect, the present invention provides a formulation of a
long-acting human growth hormone conjugate, comprising a
long-acting human growth hormone (hGH) conjugate in which the human
growth hormone (hGH) as a physiologically active peptide is linked
to an immunoglobulin Fc region, a buffer, a non-ionic surfactant
and a sugar alcohol.
In another aspect, the present invention provides a lyophilized
formulation of a long-acting human growth hormone conjugate,
comprising a lyophilized mixture of an aqueous solution comprising
a long-acting hGH conjugate in which the hGH as a physiologically
active peptide is linked to an immunoglobulin Fc region and an
albumin-free solution comprising a buffer, a non-ionic surfactant
and a sugar alcohol.
In an exemplary embodiment, the buffer is an acetate buffer, a
histidine buffer or a citrate buffer.
In another exemplary embodiment, the buffer is an acetate
buffer.
In another exemplary embodiment, the acetate is sodium acetate and
the citrate is sodium citrate.
In another exemplary embodiment, the pH of the buffer ranges from
5.0 to 6.0.
In another exemplary embodiment, the sugar alcohol is mannitol or
sorbitol.
In another exemplary embodiment, the sugar alcohol is included with
a concentration ranging from 1% (w/v) to 10% (w/v) of the total
volume of the aqueous solution.
In another exemplary embodiment, the sugar alcohol is included with
a concentration ranging from 2.5% (w/v) to 5% (w/v).
In another exemplary embodiment, the non-ionic surfactant is
polysorbate 80.
In another exemplary embodiment, the concentration of the non-ionic
surfactant ranges from 0.001% (w/v) to 0.05% (w/v) of the total
volume of the aqueous solution.
In another exemplary embodiment, the albumin-free solution further
comprises at least one selected from the group consisting of a
sugar, a polyhydric alcohol and an amino acid.
In another exemplary embodiment, the amino acid is histidine or
glycine.
In another exemplary embodiment, the concentration of the histidine
ranges from 1 to 10 mM.
In another exemplary embodiment, the concentration of the
long-acting hGH conjugate ranges from 10 to 100 mg/mL.
In another exemplary embodiment, the albumin-free solution further
comprises an isotonic agent.
In another exemplary embodiment, the isotonic agent is sodium
chloride.
In another exemplary embodiment, the concentration of the sodium
chloride ranges from 0 to 200 mM.
In another exemplary embodiment, a container of the lyophilized
formulation is a vial, a dual chamber cartridge or a dual chamber
syringe.
In another aspect, the present invention provides a liquid
formulation of a long-acting hGH conjugate, comprising a
pharmaceutically effective amount of a long-acting hGH conjugate in
which the hGH as a physiologically active peptide is linked to an
immunoglobulin Fc region and an albumin-free stabilizer, wherein
the stabilizer comprises a buffer, a non-ionic surfactant and a
sugar alcohol.
In an exemplary embodiment, the liquid formulation does not
comprise an isotonic agent.
In another exemplary embodiment, the buffer is a citrate buffer, an
acetate buffer or a histidine buffer.
In another exemplary embodiment, the sugar alcohol is mannitol or
sorbitol.
In another exemplary embodiment, the sugar alcohol is included with
a concentration ranging from 2% (w/v) to 4.5% (w/v).
In another exemplary embodiment, the sugar alcohol is included with
a concentration of 4% (w/v).
In another exemplary embodiment, the pH of the buffer ranges from
5.0 to 6.0.
In another exemplary embodiment, the pH of the buffer is 5.2.
In another exemplary embodiment, the non-ionic surfactant is
polysorbate 80.
In another exemplary embodiment, the concentration of the non-ionic
surfactant ranges from 0.001% (w/v) to 0.05% (w/v) of the total
volume of the formulation.
In another exemplary embodiment, the long-acting hGH conjugate is
included in the formulation with a concentration ranging from 5.0
mg/mL to 60.0 mg/mL.
In another exemplary embodiment, the hGH has the same amino acid
sequence as that of the wild-type hGH.
In another exemplary embodiment, the immunoglobulin Fc region is an
Fc region derived from IgG, IgA, IgD, IgE or IgM.
In another exemplary embodiment, each domain of the immunoglobulin
Fc region is a hybrid of domains with different origin derived from
an immunoglobulin selected from the group consisting of IgG, IgA,
IgD, IgE and IgM.
In another exemplary embodiment, the immunoglobulin Fc region is a
dimer or a multimer composed of a single-chain immunoglobulin
consisting of domains with the same origin.
In another exemplary embodiment, the immunoglobulin Fc region is an
IgG4 Fc region.
In another exemplary embodiment, the immunoglobulin Fc region is an
aglycosylated human IgG4 Fc region.
In another exemplary embodiment, the conjugate is in such a form
that the hGH is linked to the immunoglobulin Fc via a non-peptidyl
polymer as a linker or via genetic recombination.
In another exemplary embodiment, the non-peptidyl polymer is
selected from the group consisting of a biodegradable polymer such
as polyethylene glycol, polypropylene glycol, a copolymer of
ethylene glycol and propylene glycol, a polyoxyethylated polyol,
polyvinyl alcohol, a polysaccharide, dextran, polyvinyl ethyl
ether, polylactic acid (PLA) and polylactic-glycolic acid (PLGA), a
lipid polymer, chitin, hyaluronic acid and a combination
thereof.
In another exemplary embodiment, the non-peptidyl polymer is
polyethylene glycol.
In another exemplary embodiment, the formulation is for the
treatment of pituitary dwarfism, growth hormone deficiency,
Prader-Willi syndrome or idiopathic short stature.
In another aspect, the present invention provides a lyophilized
formulation of a long-acting hGHconjugate, comprising a lyophilized
mixture of an aqueous solution comprising a long-acting hGH
conjugate in which the hGH as a physiologically active peptide is
linked to an immunoglobulin Fc region and an albumin-free solution
comprising an acetate buffer, polysorbate 80 and mannitol.
In another aspect, the present invention provides a liquid
formulation of a long-acting hGHconjugate, comprising a
pharmaceutically effective amount of a long-acting hGH conjugate in
which the hGHas a physiologically active peptide is linked to an
immunoglobulin Fc region and an albumin-free stabilizer comprising
a citrate buffer, polysorbate 80 and mannitol, the stabilizer not
comprising an isotonic agent.
In another aspect, the present invention provides a method for
preparing the lyophilized formulation, comprising lyophilizing a
long-acting hGH conjugate in which the human growth hormone as a
physiologically active peptide is linked to an immunoglobulin Fc
region and an albumin-free solution comprising a buffer, a
non-ionic surfactant and a sugar alcohol.
In another aspect, the present invention provides a method for
reconstituting the lyophilized formulation, comprising adding a
solution for reconstitution to the lyophilized mixture of an
aqueous solution comprising a long-acting hGH conjugate in which
the human growth hormone (hGH) as a physiologically active peptide
is linked to an immunoglobulin Fc region and an albumin-free
solution comprising a buffer, a non-ionic surfactant and a sugar
alcohol included in the lyophilized formulation.
In an exemplary embodiment, the solution for reconstitution is
water for injection.
In another exemplary embodiment, the solution for reconstitution
further comprises a preservative.
In another exemplary embodiment, the preservative is benzyl
alcohol, m-cresol or phenol.
In another exemplary embodiment, the formulation reconstituted by
the method comprises the long-acting hGH conjugate with a
concentration ranging from 10 to 100 mg/mL.
In another aspect, the present invention provides a kit comprising
the lyophilized formulation of a long-acting hGH conjugate.
MODE FOR INVENTION
In an aspect, the present invention provides a formulation of a
long-acting human growth hormone conjugate, comprising a
long-acting human growth hormone (hGH) conjugate in which the hGH
as a physiologically active peptide is linked to an immunoglobulin
Fc region, a buffer, a non-ionic surfactant and a sugar
alcohol.
As used herein, the term "long-acting human growth hormone (hGH)
conjugate" refers to a conjugate in which the physiologically
active peptide human growth hormone is linked to an immunoglobulin
Fc region and the physiological activity of which has an increased
in vivo duration when compared to a wild-type hGH. The term
"long-acting" as used herein means that the physiological activity
has a longer duration than a wild-type hGH. As used herein, the
term "conjugate" refers to a form in which the human growth hormone
is coupled to an immunoglobulin Fc region.
Specifically, the formulation may be a lyophilized formulation of a
long-acting hGH conjugate, comprising a lyophilized mixture of an
aqueous solution comprising a long-acting hGH conjugate in which
the hGH as a physiologically active peptide is linked to an
immunoglobulin Fc region and an albumin-free solution comprising a
buffer, a non-ionic surfactant and a sugar alcohol.
As used herein, the term "lyophilized formulation of a long-acting
hGH conjugate" refers to a lyophilized formulation comprising a
long-acting hGH conjugate. It includes a formulation comprising
materials existing in solid state, obtained by lyophilizing a
long-acting hGH conjugate and a substance to stabilize the same
such as an excipient. In the present invention, the lyophilized
formulation includes the lyophilized substance itself. The
lyophilized substance may also be referred to as a lyophilized
cake.
The lyophilized formulation is prepared by a lyophilization process
of sublimating water from a preparatory formulation comprising a
long-acting hGH conjugate and an excipient for stabilizing the
long-acting hGH conjugate. In the present invention, the
lyophilized formulation of a long-acting hGH conjugate may comprise
a therapeutically effective amount of a long-acting hGH conjugate
and a therapeutically effective amount of the hGH may be contained
in a single-use container or a multi-use container, although being
not limited thereto.
The lyophilized formulation may be contained in a vial (e.g., a
reinforced glass vial), a dual chamber cartridge or a dual chamber
syringe, although being not limited thereto.
The lyophilized formulation of the present invention has a
composition capable of stabilizing the long-acting hGH conjugate
during a lyophilization process and capable of maintaining the
stability of the formulation when it is reconstituted after
storage. In particular, the lyophilized formulation of the present
invention is capable of providing stability even when the
long-acting hGH conjugate is included with a high concentration
ranging from 10 mg/mL to 100 mg/mL.
The lyophilized formulation of a long-acting hGH conjugate may be
stored in a container and reconstituted when administration to a
subject is necessary.
As used herein, the term "reconstitution" means that the
lyophilized substance in solid state liquefied to allow for
administration of the hGH conjugate. The concentration of the
long-acting hGH conjugate included in the lyophilized formulation
of the present invention ranges from 1 mg/mL to 150 mg/mL,
specifically from 10 mg/mL to 120 mg/mL, more specifically from 10
mg/mL to 100 mg/mL, upon reconstitution, although not being limited
thereto.
The reconstitution may be conducted by dissolving the lyophilized
substance by adding a solvent to a vial containing the lyophilized
substance or by adding a solvent to the lyophilized substance
contained in a single-use syringe or a multi-use syringe, but is
not specially limited thereto.
The lyophilized formulation of a long-acting hGH conjugate of the
present invention is advantageous over the existing liquid
formulation in that the hGH conjugate can be stably stored and the
effective concentration of the conjugate can be controlled. The
concentration after the reconstitution may be identical to or
different from the concentration of the pre-lyophilized formulation
during the lyophilization process.
The lyophilized formulation of the present invention comprises a
lyophilized mixture of an aqueous solution comprising a long-acting
hGHconjugate and an albumin-free solution comprising a buffer, a
non-ionic surfactant and a sugar alcohol.
As used herein, the term "albumin-free solution" refers to a
substance which is capable of allowing the long-acting hGH
conjugate to be stably stored and maintain its stability during
lyophilizing and reconstitution processes. In particular, the
albumin-free solution refers to an aqueous solution which comprises
a long-acting hGH conjugate and an excipient stabilizing the same
and, thus, provides stability of the long-acting hGH conjugate
during a lyophilization process and allows the preparation of a
lyophilized formulation having storage stability. Specifically, the
aqueous solution comprises a buffer, a sugar alcohol and a
non-ionic surfactant. Further, an isotonic agent may be included
for adjustment of osmotic pressure. For proteins such as the
long-acting hGH conjugate, storage stability is important not only
to ensure an accurate administration dosage but also to suppress
the potential formation of antigenic substances against the
long-acting hGH conjugate. In the present invention, the term
albumin-free solution may be used interchangeably with a
"preformulation".
Since the concentration of the long-acting hGH conjugate can be
controlled by adjusting the volume of the solution for
reconstitution added to the lyophilized formulation, the
concentration of the long-acting hGH conjugate in the albumin-free
solution is not particularly limited. However, the formulation of
the present invention is advantageous in that even an albumin-free
solution comprising the long-acting hGH conjugate with a high
concentration of 10-100 mg/mL or above can be stably lyophilized,
the prepared lyophilized formulation can be quickly dissolved
within 3 minutes and the stability of the long-acting hGH conjugate
can be maintained in the reconstituted solution.
The aqueous solution does not contain human serum albumin. Since
the human serum albumin that can be used as a protein stabilizer is
produced from human serum, there is a risk of contamination by
pathogenic viruses derived from human. In addition, gelatin or
bovine serum albumin may cause diseases or may induce an allergic
response in some patients. Since the albumin-free solution of the
present invention proteins does not contain heterologous proteins
such as serum albumin derived from human or animal or purified
gelatin, there is no risk of viral infection.
As used herein, the term "buffer" refers to a solution that is
comprised in the albumin-free solution of the present invention and
works to maintain a stable pH level of the formulation after a
lyophilization or reconstitution process such that a sharp change
in pH of the formulation is prevented to keep the activity of the
long-acting hGH conjugate stable. The buffer may include an
alkaline salt (e.g., sodium or potassium phosphate, or monobasic or
dibasic salts thereof), a citrate (e.g., sodium citrate or citric
acid), an acetate (e.g., sodium acetate or acetic acid), histidine,
any other pharmaceutically acceptable pH buffering agent known in
the art, or a combination thereof. The buffer may be specifically
an acetate buffer, a histidine buffer or a citrate buffer, more
specifically an acetate buffer or a citrate buffer, although not
being limited thereto.
The concentration of the citrate or acetate that constitutes the
buffer is specifically in a range from 5 to 100 mM, more
specifically in a range from 10 to 50 mM, although not being
limited thereto. The pH of the buffer is specifically in a range
from 4.0 to 7.0, more specifically in a range from 5.0 to 6.0,
further more specifically in a range from 5.2 to 6.0, although not
being limited thereto.
As used herein, the term "sugar alcohol" refers to a hydrogenated
carbohydrate that is comprised in the lyophilized formulation of
the present invention and works to protect the protein of the
long-acting hGH conjugate during a lyophilization process and to
improve the stability of the long-acting hGH conjugate after
reconstitution. The concentration of the sugar alcohol used in the
present invention may be in a range from 1 to 10% (w/v) of the
total volume of the formulation, although not being limited
thereto. Specifically, the concentration of the sugar alcohol may
be in a range from 2.5 to 5% (w/v). When the concentration of the
sugar alcohol is within this range, a reconstituted formulation
obtained by reconstitution using a solution for reconstitution of
the same volume as that of the preformulation may have an osmotic
pressure corresponding to that of an isotonic solution, although
not being limited thereto.
The sugar alcohol used in the present invention may be at least one
selected from the group consisting of mannitol and sorbitol,
specifically mannitol, but is not specially limited thereto. Being
included in the formulation of the present invention, the sugar
alcohol such as mannitol may serve to adjust osmotic pressure.
Specifically, the formulation obtained by reconstituting the
lyophilized formulation of the present invention may be isotonic.
However, it a hypertonic or hypotonic formulation is also suitable
in the present invention.
As used herein, the term "non-ionic surfactant" refers to a
substance that reduces the surface tension of a protein solution to
prevent the protein from being adsorbed onto a hydrophobic surface
or from aggregating after reconstitution. Specific examples of the
non-ionic surfactant that can be used in the present invention
include a polysorbate-type non-ionic surfactant, a poloxamer-type
non-ionic surfactant and a combination thereof. More specifically,
a polysorbate-type non-ionic surfactant may be used. Examples of
the polysorbate-type non-ionic surfactant include polysorbate 20,
polysorbate 40, polysorbate 60 and polysorbate 80, and among them
polysorbate 80 is preferred, although not being limited thereto. It
may not be appropriate to add the non-ionic surfactant at a high
concentration to the formulation, since a non-ionic surfactant at
high concentration can cause interference effects when protein is
analyzed to determine protein concentration or stability through
analytic methods such as UV-spectrometric method or isoelectric
focusing (IEF) and thus make it hard to determine the protein
stability accurately. Therefore, the lyophilized formulation of the
present invention may comprise the non-ionic surfactant
specifically at a low concentration of 0.1% (w/v) or less, more
specifically in a range from 0.001 to 0.1% (w/v), and further more
specifically in a range from 0.001 to 0.05% (w/v), although not
being limited thereto.
Specifically, the albumin-free solution may further comprise at
least one selected from the group consisting of a sugar, a
polyhydric alcohol and an amino acid. It was confirmed by the
inventors of the present invention that, when histidine is further
added as the amino acid, dissolution rate can be improved and
reconstitution can be achieved without foaming.
Specific examples of the sugar that can be further included to
increase the storage stability of the long-acting hGH conjugate
include monosaccharides such as mannose, glucose, fucose and xylose
and polysaccharides such as lactose, maltose, sucrose, raffinose
and dextran. Specific examples of the polyalcohol include propylene
glycol, low-molecular-weight polyethylene glycol, glycerol,
low-molecular-weight polypropylene glycol and a combination
thereof. Examples of the amino acid include histidine or glycine,
although not being limited thereto. The histidine, etc. may be
present in the aqueous solution at a concentration ranging from 1
to 10 mM, although not being limited thereto.
The albumin-free solution may further comprise an isotonic agent
for control of osmotic pressure.
As used herein, the term "isotonic agent" refers to an agent that
maintains an appropriate osmotic pressure when the long-acting hGH
conjugate is administered into the body after being reconstituted.
The isotonic agent may have an effect of further stabilizing the
long-acting hGH conjugate in solution. For example, the isotonic
agent may be a water-soluble inorganic salt, specifically sodium
chloride, although not being limited thereto. The concentration of
sodium chloride used in the present invention is specifically in a
range from 0 to 200 mM, although not being limited thereto. Also,
depending on the type and amount of the substances comprised in the
formulation, the amount of the isotonic agent included can be
adjusted such that the solution formulation comprising all of the
ingredients becomes isotonic.
The albumin-free solution may be lyophilized after being diluted
1/2-fold, 1/4-fold or further. It was confirmed by the inventors of
the present invention that dissolution time can be reduced when the
albumin-free solution is lyophilized after being diluted 1/2-fold
or 1/4-fold (Test Example 1-(5)).
The lyophilized formulation of the present invention comprising the
lyophilized mixture exhibits superior dissolution time. The
dissolution time is one of important properties of a lyophilized
substance. If the dissolution time is long, the protein has to be
exposed to a concentrated solution for a long time, during which it
can be denatured. Also, since the incompletely dissolved product
cannot be administered, a short dissolution time can provide
convenience for both the patients and the physicians. However, the
dissolution time is inevitably increased as the protein
concentration increases. Accordingly, development of a formulation
with short dissolution time can be an important issue for a
high-concentration lyophilized formulation.
It was confirmed by the inventors of the present invention that the
lyophilized formulation of the present invention is dissolved
within as short as 10 seconds and as long as 3 minutes even when it
comprised the long-acting hGH conjugate at a high concentration of
10 mg/mL or above.
The desired dosage of a lyophilized product may be achieved by
lyophilizing the target protein with a desired concentration and
then reconstituting the same with a volume of a preformulation,
although not being specially limited thereto. Alternatively, the
preformulation may be lyophilized with an increased volume through
dilution and then reconstituted using a solution for reconstitution
with a smaller volume. However, if the preformulation is overly
diluted, the lyophilization cycle (in particular, primary drying
time) may be prolonged and thus production cost may be increased.
Accordingly, the formulation of the present invention is also
advantageous in terms of cost in that even the high-concentration
long-acting hGH conjugate can be lyophilized without excessive
dilution.
In addition, the lyophilized formulation of the present invention
may further comprise other ingredients or materials that are known
in the art in addition to the above-described buffer, isotonic
agent, sugar alcohol, non-ionic surfactants and the preservative
included in the solution for reconstitution, unless they do not
diminish the effect of the present invention.
The inventors of the present invention prepared, as a
preformulation, a lyophilized formulation comprising a long-acting
hGH conjugate and an albumin-free solution comprising a buffer, a
sugar alcohol and a surfactant and evaluated its stability and
dissolution rate. Specifically, a long-acting hGH conjugate was
lyophilized with a concentration of 19.5 mg/mL or 78.0 mg/mL using
a pre-lyophilized formulation comprising a 20 mM citrate buffer of
pH 5.2 or pH 5.6, 150 mM sodium chloride, 5% mannitol and 0.005%
polysorbate 80 and then reconstituted using distilled water.
Superior stability was superior at the above concentrations. In
particular, the stability was better at the higher concentration of
78.0 mg/mL. Also, good stability was achieved at pH 5.2 and 5.6
both (Test Example 1-(1)). In addition, when a long-acting hGH
conjugate was dissolved in a pre-lyophilized formulation comprising
a 20 mM acetate buffer of pH 5.2 or pH 5.6, 150 mM NaCl, 5%
mannitol and 0.005% polysorbate 80 and then reconstituted using a
solution for reconstitution comprising m-cresol, benzyl alcohol or
phenol as a preservative, the stability was maintained. This result
confirms that a reconstituted formulation with preserved activity
can be prepared by reconstituting a lyophilized substance with a
solution for reconstitution comprising a preservative. In
particular, when the aqueous acetate solution was used, superior
stability could be achieved without precipitation even when the
reconstitution was conducted using a solution for reconstitution
comprising a preservative (Test Example 1-(2)). Furthermore, it was
confirmed that the dissolution time of the prepared lyophilized
formulation is increased as the concentration of the conjugate
increases from 19.5 mg/mL to 39.0 mg/mL to 58.5 mg/mL and to 70.0
mg/mL (Test Example 1-(3)). In addition, superior dissolution time
and stability were achieved even when the albumin-free solution did
not contain a salt and the dissolution rate could be increased by
increasing the concentration of mannitol. And, when histidine was
added, it was possible to reduce the dissolution time without
increasing the concentration of mannitol (Test Example 1-(4)).
Further, the inventors found out that the dissolution rate is
further improved when the preformulation is diluted to decrease
density (Test Example 1-(5)) and established the optimized drying
condition under which the lyophilized substance can be completely
dried (Test Example 1-(6)). It was also demonstrated that the
presence of mannitol greatly affects the dissolution time (Test
Example 1-(7)) and that an isotonic osmotic pressure can be
achieved with a dissolution time of 30 seconds or less when the
mannitol is concentration 4-4.5% even for the high-concentration
long-acting hGH conjugate of 58.5 mg/mL (Test Example 1-(8)). In
addition, the stability was maintained nearly constant even after
the prepared lyophilized formulations were stored at 4.degree. C.
or 25.degree. C. for 6 months. Also, the stability was maintained
even after the lyophilized formulations were stored at 25.degree.
C. for 2 weeks after reconstitution (Test Example 1-(9)).
Specifically, the formulation may be a liquid formulation of a
long-acting hGH conjugate, comprising a long-acting hGH conjugate
in which the hGH as a physiologically active peptide is linked to
an immunoglobulin Fc region and an albumin-free stabilizer, wherein
the stabilizer comprises a buffer, a non-ionic surfactant and a
sugar alcohol.
The long-acting hGH conjugate is the same as described above.
As used herein, the term "liquid formulation of a long-acting hGH
conjugate" refers to a liquid formulation which comprises a
long-acting hGH conjugate. The liquid formulation includes liquid
formulations for both internal and external application. In the
present invention, the liquid formulation of a long-acting hGH
conjugate may comprise a pharmaceutically effective amount of the
long-acting hGH conjugate. In general, the pharmaceutically
effective amount of hGH corresponds to about 1 to 3 mg in a
single-use vial, but is not limited thereto.
And, the concentration of the long-acting hGH conjugate comprised
in the liquid formulation of the present invention ranges
specifically from 5.0 to 60.0 mg/mL, although not being limited
thereto.
The liquid formulation of a long-acting hGH conjugate comprises a
pharmaceutically effective amount of a long-acting hGH conjugate
and an albumin-free stabilizer.
As used herein, the term "stabilizer" refers to a substance that
allows the long-acting hGH conjugate to be stored stably.
Specifically, the stabilizer comprises a buffer, a sugar alcohol
and a non-ionic surfactant. With regard to proteins such as the
long-acting hGH conjugate, the storage stability is important for
ensuring dose accuracy and suppressing the formation of potential
antigens against the long-acting hGH conjugate.
As used herein, the term "buffer" refers to a solution that is
comprised in the stabilizer of the present invention and works to
maintain a stable pH level of the formulation, thereby preventing a
drastic change in pH to maintain the activity of the long-acting
hGH conjugate stable. The description of the buffer stated above
with regard to the lyophilized formulation also applies here.
The buffer may be a citrate buffer, an acetate buffer or a
histidine buffer, specifically a citrate buffer, although not being
limited thereto. The concentration of the salt that constitutes the
buffer is specifically in a range from 5 to 100 mM, more
specifically in a range from 10 to 50 mM, although not being
limited thereto. The pH of the buffer is in a range from 4.0 to
7.0, more specifically in a range from 5.0 to 6.0, further more
specifically in a range from 5.2 to 6.0, most specifically 5.2,
although not being limited thereto.
As used herein, the term "sugar alcohol" refers to a hydrogenated
carbohydrate that is comprised in the liquid formulation of the
present invention and works to improve the stability of the
long-acting hGH conjugate. The concentration of the sugar alcohol
used in the present invention is specifically in a range from 1 to
10% (w/v) of a total volume of the formulation, and more
specifically in a range from 2% (w/v) to 4.5% (w/v), further more
specifically 4% (w/v), although not being limited thereto. The
sugar alcohol used in the present invention may be at least one
selected from the group consisting of mannitol and sorbitol,
specifically mannitol, although not specially limited thereto.
As used herein, the term "non-ionic surfactant" refers to a
substance that reduces the surface tension of a protein solution to
prevent the protein from being adsorbed onto a hydrophobic surface
or from aggregating. Specific examples of the non-ionic surfactant
that can be used in the present invention are the same as described
above.
The formulation of the present invention may be one not containing
an isotonic agent.
The isotonic agent is the same as described above.
The inventors of the present invention found that a formulation
comprising an aqueous acetate or citrate solution of pH 5.2 as a
buffer and comprising 4% (w/v) mannitol but not comprising an
isotonic agent exhibits superior stability. In particular, a
formulation comprising an aqueous citrate solution of pH 5.2 as a
buffer and comprising 4% (w/v) mannitol but not comprising an
isotonic agent exhibited the highest stability at the long-acting
hGH conjugate concentration of 10.0 mg/mL.
Specific examples of the sugar that can be further included to
increase the storage stability of the long-acting hGH conjugate are
the same as described above. In addition, the liquid formulation of
the present invention may further comprise other ingredients or
materials that are known in the art in addition to the
above-described buffer, sugar alcohol and non-ionic surfactants,
unless they do not diminish the effect of the present
invention.
In particular, the liquid formulation of the present invention may
further comprise a preservative.
It was found that a formulation of a long-acting hGH conjugate not
containing an isotonic agent exhibits the best stability of the
long-acting hGH conjugate in a buffer at pH 5.2 when it comprises
4% mannitol (w/v). In particular, the best stability was achieved
at the long-acting hGH conjugate concentration of 10.0 mg/mL when
an acetate buffer was used (Test Example 2).
The formulation of the present invention may be for the treatment
of pituitary dwarfism, growth hormone deficiency, Prader-Willi
syndrome or idiopathic short stature and may be injected into after
reconstitution to treat the diseases.
Hereinafter, the long-acting hGH conjugate is described in
detail.
As used herein, the term "human growth hormone (hGH)" refers to a
peptide hormone that stimulates growth, cell reproduction and
regeneration in humans. The information on the sequence of the hGH
can be obtained from common database such as the NCBI GenBank. In
addition, the scope of the hGH in the present invention includes a
protein possessing an amino acid sequence having a sequence
homology of 70% or higher, specifically 80% or higher, more
specifically 90% or higher, even more specifically 95% or higher,
and most specifically 98% or higher to an amino acid sequence of a
wild-type hGH, as long as it has a hGH activity. Also, as long as
its biological activity is not significantly changed, any mutant
derived from a wild-type hGH by substitution, deletion, or
insertion of amino acid residues may be used in the present
invention.
The hGH useful in the present invention may have an amino acid
sequence of a wild-type hGH, its variant, its derivative, or
fragments thereof.
As used herein, the term "hGH variant" refers to a peptide having
at least one amino acid sequences different from those of the
wild-type hGH while demonstrating the hGH activity. The hGH variant
may be prepared by substitution, addition, deletion, or
modification of some amino acids of the wild-type hGH or a
combination thereof.
As used herein, the term "hGH derivative" refers to a peptide
having at least 80% amino acid sequence homology to the wild-type
hGH and exhibiting the hGH activity, in which some groups of the
amino acid residues are chemically substituted (e.g.,
alpha-methylation, alpha-hydroxylation), deleted (e.g.,
deamination), or modified (e.g., N-methylation).
As used herein, the term "hGH fragment" refers to a peptide in
which at least one amino acids are added or deleted at the
N-terminal or the C-terminal of the hGH while retaining the hGH
activity. The added amino acid be one which does not naturally
occur (for example, D-amino acid).
In addition, the hGH used in the present invention may be obtained
from a native or recombinant protein. Specifically, it is the
recombinant hGH prepared by using E. coli as a host cell, although
not being limited thereto.
As used herein, the term "immunoglobulin Fc region" refers to a
part of immunoglobulin excluding the variable regions of the heavy
chain and light chain, the heavy-chain constant region 1 (C.sub.H1)
and the light-chain constant region 1 (C.sub.L1) of the
immunoglobulin. The immunoglobulin Fc region may be the heavy-chain
constant region 2 (C.sub.H2) and the heavy-chain constant region 3
(C.sub.H3) of an immunoglobulin, and may further comprise a hinge
region at the heavy-chain constant region, although not being
limited thereto. Also, the immunoglobulin Fc region of the present
invention may be an extended Fc region that comprises a portion or
full of the heavy-chain constant region 1 (C.sub.H1) and/or the
light-chain constant region 1 (C.sub.L1) except for the variable
regions of the heavy chain and light chain of immunoglobulin, as
long as it has substantially the same or improved effect as
compared to the wild-type protein. Also, the immunoglobulin Fc
region may be a fragment wherein a considerably long portion of the
amino acid sequence corresponding to C.sub.H2 and/or C.sub.H3 is
deleted. That is, the immunoglobulin Fc region of the present
invention may comprise 1) a C.sub.H1 domain, a C.sub.H2 domain, a
C.sub.H3 domain and a C.sub.H4 domain, 2) a C.sub.H1 domain and a
C.sub.H2 domain, 3) a C.sub.H1 domain and a C.sub.H3 domain, 4) a
C.sub.H2 domain and a C.sub.H3 domain, 5) a combination of at least
one domains and an immunoglobulin hinge region (or a portion of the
hinge region), and 6) a dimer of a domain of the heavy-chain
constant regions and a light-chain constant region, although not
being limited thereto.
The immunoglobulin Fc region of the present invention comprises a
native amino acid sequence and an amino acid sequence derivative
(mutant) thereof. The amino acid sequence derivative refers to the
sequence having different sequence from the native sequence by
deletion, insertion, non-conservative or conservative substitution
of at least one amino acid residues of the native amino acid
sequence, or combinations thereof. For example, in IgG Fc, amino
acid residues at positions 214 to 238, 297 to 299, 318 to 322, or
327 to 331 which are known to be important for protein binding may
be suitable targets for modification.
Also, other types of derivatives may be used including the
derivatives wherein a region capable of forming a disulfide bond is
deleted, few amino acid residues at the N-terminal of a native Fc
are eliminated, or a methionine residue is added at the N-terminal
of the native Fc. Further, in order to eliminate the function of
effector, a complement-binding site, for example C1q-binding site
or antibody dependent cell mediated cytotoxicity (ADCC) site, may
be deleted. Techniques for preparing such sequence derivatives of
the immunoglobulin Fc region are disclosed in WO 97/34631 and WO
96/32478.
Substitution of amino acids in proteins and peptides, which do not
change the overall protein activities, are known in the art (H.
Neurath, R. L. Hill, The Proteins, Academic Press, New York, 1979).
The most-commonly occurring exchanges are Ala/Ser, Val/Ile,
Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly,
Thy/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu and
Asp/Gly, in both directions. In some cases, the Fc region may be
modified by phosphorylation, sulfation, acrylation, glycosylation,
methylation, farnesylation, acetylation, amidation, and the like.
The aforementioned Fc derivatives demonstrate the same biological
activity as the Fc region of the present invention, and they have
an enhanced structural stability against heat, pH, and the
like.
In addition, these Fc regions may be obtained from native proteins
isolated from humans or other animals including cows, goats, swine,
mice, rabbits, hamsters, rats and guinea pigs, or may be
recombinants obtained from transformed animal cells or
microorganisms or derivatives thereof. Here, the method of
obtaining Fc regions from native immunoglobulin may include
isolating the whole immunoglobulins from human or animal bodies and
treating them with a protease. When papain is used for digesting
immunoglobulins, they are cleaved into Fab and Fc regions, and when
pepsin is used, the immunoglobulin is cleaved into pF'c and
F(ab).sub.2. These fragments may be separated by size exclusion
chromatography to isolate Fc or pF'c. Specifically, a human-derived
Fc region is a recombinant immunoglobulin Fc region obtained from a
microorganism.
In addition, the immunoglobulin Fc region of the present invention
may be in a form of native sugar chains, longer sugar chains than
native form, shorter sugar chains than native form, or a
deglycosylated form. The extension or removal of the immunoglobulin
Fc sugar chains may be done by using common methods in the art
including chemical methods, enzymatic methods, and gene engineering
method using a microorganism. The removal of sugar chains from an
immunoglobulin Fc region results in a drastic decrease in its
binding affinity to C1q of the first complement component C1 and
thus antibody-dependent cell-mediated cytotoxicity or
complement-dependent cytotoxicity is reduced or removed, and the
occurrence of unnecessary immune responses in vivo can be avoided.
In this regard, a deglycosylated or aglycosylated immunoglobulin Fc
region is more preferable form as a drug carrier for the object of
the present invention.
In addition, the immunoglobulin Fc region may be one derived from
IgG, IgA, IgD, IgE and IgM, or those prepared by a combination or
hybrid thereof. Specifically, it is derived from IgG or IgM, which
are among the most abundant proteins in human blood, and most
specifically from IgG, which is known to enhance the half-life of a
ligand-binding protein. The immunoglobulin Fc may be generated by
treating a native IgG with a certain protease, or by transformed
cells using the genetic recombination technique. Specifically, the
immunoglobulin Fc is a recombinant human immunoglobulin Fc produced
in E. coli.
Meanwhile, the term "combination", as used herein, refers to a
conjugation between a polypeptide encoding single-chain
immunoglobulin Fc regions of the same origin and a single-chain
polypeptide of different origin when forming a dimer or multimer.
That is, a dimer or multimer can be formed from two or more
fragments selected from the group consisting of IgG Fc, IgA Fc, IgM
Fc, IgD Fc and IgE Fc fragments.
As used herein, the term "hybrid" refers to the presence of at
least two sequences corresponding to immunoglobulin Fc fragments of
different origins in a single-chain immunoglobulin Fc region. In
the present invention, various types of hybrids may be used. That
is, a hybrid of domains may be composed of one to four domains
selected from the group consisting of C.sub.H1, C.sub.H2, C.sub.H3
and C.sub.H4 of IgG Fc, IgM Fc, IgA Fc, IgE Fc and IgD Fc, and may
comprise a hinge region.
Meanwhile, IgG may also be divided into subclasses, IgG1, IgG2,
IgG3 and IgG4, and a combination or hybrid thereof is also possible
in the present invention, specifically IgG2 and IgG4 subclasses,
and most specifically Fc region of IgG4 that lacks an effector
function such as complement-dependent cytotoxicity. In other words,
the most preferable immunoglobulin Fc region of the conjugate in
the present invention is a human IgG4-derived non-glycosylated Fc
region. The human-derived Fc region is preferred to a non-human
derived Fc region which can act as an antigen in human body and
cause undesirable immune responses such as production of new
antibodies against the antigen.
The long-acting hGH conjugate of the present invention can be
prepared by combining a hGH prepared from a native or recombinant
form by any method and an immunoglobulin Fc region prepared by
treating a wild-type IgG with a certain protease or produced from a
transformed cell by using the recombination technique.
As a combining method used for this purpose, the conjugate can be
prepared by cross-linking the hGH and the immunoglobulin Fc region
using a non-peptidyl polymer or can be produced as a fusion protein
wherein the hGH and the immunoglobulin Fc region are linked using
the recombination technique. That is, the conjugate can be produced
in a form where the hGH and the immunoglobulin Fc are linked via a
non-peptidyl linker, or in a form of a fusion protein of the hGH
and the immunoglobulin Fc. The fusion protein comprises a form
where the hGH and the immunoglobulin Fc are combined via a peptidyl
linker, although not being limited thereto.
As used herein, the term "non-peptidyl polymer" refers to a
biocompatible polymer in which two or more repeating units are
combined and the repeating units are connected to each other by any
covalent bonding except for peptide bonding. In the present
invention, the term non-peptidyl polymer may be used
interchangeably with the term non-peptidyl linker.
The non-peptidyl polymer used for cross-linking may be selected
from the group consisting of a biodegradable polymer including
polyethylene glycol, polypropylene glycol, an ethylene
glycol-propylene glycol copolymer, polyoxyethylated polyol,
polyvinyl alcohol, polysaccharide, dextran, polyvinyl ethyl ether,
polylactic acid (PLA) or polylactic-glycolic acid (PLGA), a lipid
polymer, chitin, hyaluronic acid and a combination thereof.
Specifically, polyethylene glycol (PEG) may be used, although not
being limited thereto. In addition, their derivatives that are
already known in the art and derivatives that can be easily
prepared by a method known in the art may be included in the scope
of the present invention.
For preparation of the long-acting hGH conjugate of the present
invention, references such as Korean Patent No. 0725315 are
disclosed in the present invention as cited references. Those
skilled in the art can produce the long-acting hGH conjugate of the
present invention by consulting the references, although not being
limited thereto.
In another aspect, the present invention provides a lyophilized
formulation of a long-acting hGH conjugate, comprising a
lyophilized mixture of an aqueous solution comprising a long-acting
hGH conjugate in which the hGH as a physiologically active peptide
is linked to an immunoglobulin Fc region and an albumin-free
solution comprising an acetate buffer, polysorbate 80 and
mannitol.
The hGH, the immunoglobulin Fc region, the long-acting hGH
conjugate, the albumin-free solution, the lyophilizing and the
lyophilized formulation are the same as descried above.
In another aspect, the present invention provides a liquid
formulation of a long-acting hGHconjugate, comprising a
pharmaceutically effective amount of a long-acting hGHconjugate in
which the hGHas a physiologically active peptide is linked to an
immunoglobulin Fc region and an albumin-free stabilizer comprising
a citrate buffer, polysorbate 80 and mannitol, the stabilizer not
comprising an isotonic agent.
The hGH, the immunoglobulin Fc region, the long-acting
hGHconjugate, the albumin-free stabilizer and the liquid
formulation are the same as descried above.
In another aspect, the present invention provides a method for
preparing the lyophilized formulation, comprising lyophilizing a
long-acting hGH conjugate and an albumin-free solution comprising a
buffer, a non-ionic surfactant and a sugar alcohol.
The long-acting hGH conjugate, the buffer, the non-ionic
surfactant, the sugar alcohol, the albumin-free solution, the
lyophilization and the lyophilized formulation are the same as
descried above.
In another aspect, the present invention provides a method for
reconstituting the lyophilized formulation, comprising adding a
solution for reconstitution to the lyophilized formulation.
The lyophilized formulation and the reconstitution are the same as
descried above.
As used herein, the term "solution for reconstitution" refers to a
solution which is added to a lyophilized substance in solid state
to reconstitute the same. The solution for reconstitution may be
water for injection, e.g. sterilized distilled water, but is not
specially limited thereto.
Also, the solution for reconstitution may further comprise a
preservative.
As used herein, the term "preservative" refers to a substance that
substantially reduces bacterial or fungal contamination in a
formulation. Especially, it is comprised in the formulation to
facilitate the production of a formulation for multiple dosing.
Examples of preservative include octadecyl dimethyl benzyl ammonium
chloride, hexamethonium chloride, benzalkonium chloride (mixture of
alkylbenzyldimethylammonium chloride which has a long alkyl chain),
and benzethonium chloride. Other types of preservatives include:
aromatic alcohols such as phenol, butyl alcohol and benzyl alcohol;
alkyl paraben such as methylparaben or propylparaben; catechol;
resorcinol; cyclohexanol; 3-pentanol; and m-cresol, but are not
limited thereto. The preservative in the liquid formulation of the
present invention is specifically benzyl alcohol, m-cresol or
phenol, more specifically benzyl alcohol, although not being
limited thereto. The concentration of the preservative is
specifically in a range from 0.001 to 0.9% (w/v), more specifically
in a range from 0.1 to 0.9% (w/v), although not being limited
thereto.
The formulation of the present invention reconstituted as described
above may contain the long-acting hGH conjugate with a
concentration ranging from 10 to 100 mg/mL, although not being
limited thereto.
In another aspect, the present invention provides a kit comprising
the lyophilized formulation and a solution for reconstitution.
The lyophilized formulation is the same as described above.
The kit comprises the lyophilized formulation and a solution for
reconstitution and may further comprise a composition, solution or
apparatus comprised of at least one other ingredient suitable for
the reconstitution.
Hereinafter, the present invention is described in more detail with
reference to examples. However, these examples are for illustrative
purposes only, and the invention is not intended to be limited by
these examples.
Preparation Example
Preparation of Long-Acting Human Growth Hormone (hGH) Conjugate
ALD-PEG-ALD, which is a polyethylene glycol with a molecular weight
of about 3.4 kDa having aldehyde groups at both ends, was
conjugated with the human growth hormone (hGH, molecular weight: 22
kDa), and then linked to the N-terminal of a human IgG4-derived
aglycosylated Fc region (about kDa). Through this, the final
product hGH-PEG-Fc conjugate (hereinafter, referred to as
"long-acting hGH conjugate") which is a representative long-acting
hGH conjugate of the present invention was prepared and
purified.
Test Example 1
Evaluation of Lyophilized Formulation of Long-Acting hGH
Conjugate
(1) Analysis of Stability of Lyophilized Formulation of Long-Acting
hGH Conjugate Depending on Concentration and Buffer
After preparing lyophilized formulations comprising the long-acting
hGH conjugate at concentrations described in Table 1, stability was
analyzed after reconstitution. The effect of buffer and pH on the
stability of the long-acting hGH conjugate was also analyzed.
As described in Table 1, the long-acting hGH conjugate was
lyophilized with the given concentration using a pre-lyophilized
formulation comprising a buffer, sodium chloride (NaCl), mannitol
and polysorbate 80, which was then reconstituted using distilled
water. The lyophilizing consisted of primary drying and secondary
drying steps. The temperature gradient of the lyophilizing was set
as freezing followed by primary drying (4.degree. C.) and secondary
drying (20.degree. C.), as shown in FIG. 1. The reconstitution was
conducted by dissolving the lyophilized formulation with distilled
water of the same volume as that of the formulation before the
lyophilization. The reconstituted liquid formulation was stored at
40.degree. C. for 4 weeks and the stability was analyzed by ion
exchange chromatography (IE-HPLC). The result is shown in Table 2.
In Table 2, IE-HPLC (%) indicates the purity of the long-acting hGH
conjugate at the given time.
TABLE-US-00001 TABLE 1 Conc. Buffer Salt Sugar alcohol Surfactant
Example 19.5 mg/mL 20 mM 150 mM 5% mannitol 0.005% 1 sodium NaCl
polysorbate (Ex. 1) citrate 80 (pH 5.2) Ex. 2 78.0 mg/mL 20 mM 150
mM 5% mannitol 0.005% sodium NaCl polysorbate citrate 80 (pH 5.2)
Ex. 3 78.0 mg/mL 20 mM 150 mM 5% mannitol 0.005% sodium NaCl
polysorbate acetate 80 (pH 5.6)
TABLE-US-00002 TABLE 2 IE-HPLC (%) Week 0 Week 1 Week 2 Week 4
Example 1 96.2 93.6 87.1 76.8 Example 2 96.4 95.9 92.8 82.0 Example
3 96.4 93.6 88.8 82.2
As seen from Table 2, the liquid formulation of a long-acting hGH
conjugate showed no difference in stability depending on
concentration after being stored at 40.degree. C. for 4 weeks.
Accordingly, it was confirmed that stability can be provided even
to the long-acting hGH at high concentration. Also, it was
confirmed that the stability is maintained when the buffer and the
pH were changed (compare Examples 2 and 3).
(2) Analysis of Stability and Solubility of Lyophilized Formulation
of Long-Acting Human Growth Hormone (hGH) Conjugate Depending on
Preservative
Using the formulations of Example 2 (20 mM sodium citrate, pH 5.2,
150 mM sodium chloride, 5% mannitol, 0.005% polysorbate 80) and
Example 3 (20 mM sodium acetate, pH 5.6, 150 mM sodium chloride, 5%
mannitol, 0.005% polysorbate 80) of Test Example 1-(1) and an
isotonic formulation prepared from the formulation of Example 3 (20
mM sodium acetate, pH 5.6, 4% mannitol, 0.005% polysorbate 80), the
long-acting hGH conjugate was mixed at concentrations of 68.25
mg/mL and 58.5 mg/mL as described in Table 3, which were then
lyophilized. After reconstituting using the solution for
reconstitutions comprising preservatives described in Table 3,
dissolution time and stability were measured. The product state was
compared with unaided eyes. The lyophilizing and reconstitution
were conducted in the same manner as described in Test Example
1-(1). The reconstituted liquid formulation was stored at
25.degree. C. for 4 weeks and then stability was evaluated by ion
exchange chromatography (IE-HPLC) and visual inspection. The result
is shown in Table 4. In Table 4, IE-HPLC (%) indicates the purity
of the long-acting hGH conjugate at the given time.
TABLE-US-00003 TABLE 3 Sugar Surfac- Preser- Conc. Buffer Salt
alcohol tant vative Ex. 4 68.25 20 mM 150 mM 5% 0.005% 0.3% mg/mL
sodium NaCl mannitol polysor- m-cresol citrate bate 80 (pH 5.2) Ex.
5 68.25 20 mM 150 mM 5% 0.005% 0.9% mg/mL sodium NaCl mannitol
polysor- benzyl citrate bate 80 alcohol (pH 5.2) Ex. 6 68.25 20 mM
150 mM 5% 0.005% 0.3% mg/mL sodium NaCl mannitol polysor- m-cresol
acetate bate 80 (pH 5.6) Ex. 7 68.25 20 mM 150 mM 5% 0.005% 0.9%
mg/mL sodium NaCl mannitol polysor- benzyl acetate bate 80 alcohol
(pH 5.6) Ex. 8 58.5 20 mM -- 4% 0.005% 0.3% mg/mL sodium mannitol
polysor- m-cresol acetate bate 80 (pH 5.6) Ex. 9 58.5 20 mM -- 4%
0.005% 0.3% mg/mL sodium mannitol polysor- phenol acetate bate 80
(pH 5.6)
TABLE-US-00004 TABLE 4 IE-HPLC (%) Week 0 Week 1 Week 2 Week 4
Remarks Example 4 96.7 93.2 90.0 83.7 Precipitation occurred on
week 3 Example 5 96.7 92.6 89.8 83.1 Precipitation occurred on week
3 Example 6 96.6 93.1 89.4 87.7 -- Example 7 96.5 93.1 89.6 87.5 --
Example 8 97.6 95.4 92.3 88.1 -- Example 9 97.4 95.3 92.4 87.4
--
As seen from Table 4, the stability of the long-acting hGH
conjugate was maintained better in Examples 6 and 7 than in
Examples 4 and 5. However, as can be seen from the results for
Examples 6-9, there was no difference in the stability of the
long-acting hGH conjugate depending on the kind of the
preservatives. But, when the solution for reconstitution containing
m-cresol was used, the resulting liquid formulation was hazy during
the dissolution.
(3) Analysis of Solubility of Lyophilized Formulation of
Long-Acting hGH Conjugate Depending on Conjugate Concentration
Lyophilized formulations comprising the long-acting hGH conjugate
at different concentrations were prepared and their product state
and solubility upon reconstitution were evaluated. Using the
formulation of Example 1 (20 mM sodium citrate, pH 5.2, 150 mM
sodium chloride, 5% mannitol, 0.005% polysorbate 80) of Test
Example 1-(1), the long-acting hGH conjugate was mixed at different
concentrations as described in Table 5, which were then
lyophilized. After reconstituting using distilled water,
dissolution time was measured. The lyophilization and
reconstitution were conducted in the same manner as described in
Test Example 1-(1). The product state was compared with unaided
eyes. The reconstitution was performed using an auto shaker set to
60.degree. and 30 rpm. The time required for complete dissolution
is given in Table 6.
TABLE-US-00005 TABLE 5 Sugar Conc. Buffer Salt alcohol Surfactant
Ex. 1 19.5 mg/mL 20 mM sodium 150 mM 5% 0.005% citrate (pH 5.2)
NaCl mannitol polysorbate 80 Ex. 10 39.0 mg/mL 20 mM sodium 150 mM
5% 0.005% citrate (pH 5.2) NaCl mannitol polysorbate 80 Ex. 11 58.5
mg/mL 20 mM sodium 150 mM 5% 0.005% citrate (pH 5.2) NaCl mannitol
polysorbate 80 Ex. 12 70.0 mg/mL 20 mM sodium 150 mM 5% 0.005%
citrate (pH 5.2) NaCl mannitol polysorbate 80
TABLE-US-00006 TABLE 6 Example 1 Example 10 Example 11 Example 12
Dissolution time 10 30 90 150 (sec)
Although the product state of the lyophilized substance was stable
regardless of concentration, more rigid cakes could be observed at
higher concentrations. As seen from Table 6, the dissolution time
was increased with concentration.
(4) Analysis of Stability and Solubility of Lyophilized Formulation
of Long-Acting Human Growth Hormone (hGH) Conjugate Depending on
Stabilizer
Lyophilized formulations of the long-acting hGH conjugate were
prepared using different stabilizers and their dissolution time,
dissolution state and long-acting hGH conjugate stability were
evaluated. Preformulations were prepared with the compositions
described in Table 7, which were then used to freeze-dry the
long-acting hGH conjugate at 78.0 mg/mL. After reconstituting using
distilled water, dissolution time was measured. The lyophilization
and reconstitution were conducted in the same manner as described
in Test Example 1-(1). The product state was compared with unaided
eyes. The reconstitution was performed using an auto shaker set to
60.degree. and 30 rpm. The time required for complete dissolution
is given in Table 8.
Also, after storing the reconstituted liquid formulation at
40.degree. C. for 4 weeks, stability was evaluated by ion exchange
chromatography (IE-HPLC). In Table 9, IE-HPLC (%) indicates the
residual rate of the long-acting hGH conjugate at the given time
relative to the initial value.
TABLE-US-00007 TABLE 7 Sugar alcohol and other Conc. Buffer Salt
stabilizer Surfactant Ex. 13 78.0 20 mM sodium 150 mM 5% mannitol
0.005% mg/mL citrate (pH 5.2) NaCl polysorbate 80 Ex. 14 78.0 20 mM
sodium 150 mM 5% mannitol 0.02% mg/mL citrate (pH 5.2) NaCl
polysorbate 80 Ex. 15 78.0 20 mM sodium 150 mM 10% mannitol 0.005%
mg/mL citrate (pH 5.2) NaCl polysorbate 80 Ex. 16 78.0 20 mM sodium
150 mM 2.5% mannitol 0.005% mg/mL citrate (pH 5.2) NaCl polysorbate
80 Ex. 17 78.0 20 mM sodium 10% mannitol 0.005% mg/mL citrate (pH
5.2) polysorbate 80 Ex. 18 78.0 20 mM sodium 5% mannitol 0.005%
mg/mL citrate (pH 5.2) 2% glycine polysorbate 80 Ex. 19 78.0 20 mM
sodium 5% mannitol 0.005% mg/mL citrate (pH 5.2) 5 mM histidine
polysorbate 80
TABLE-US-00008 TABLE 8 Ex. Ex. Ex. Ex. Ex. Ex. Ex. 13 14 15 16 17
18 19 Dissolution 120 120 80 180 90 130 90 time (sec)
TABLE-US-00009 TABLE 9 IE-HPLC (%) Week 0 Week 2 Week 4 Example 13
100.0 97.8 87.9 Example 14 100.0 97.8 88.3 Example 15 100.0 96.3
85.8 Example 16 100.0 98.1 86.8 Example 17 100.0 94.7 82.6 Example
18 100.0 96.3 81.6 Example 19 100.0 95.4 83.7
As seen from Table 8, high dissolution rate was obtained when the
concentration of mannitol was high. Also, it was confirmed that the
addition of 5 mM histidine leads to improved dissolution rate. The
formulations of Examples 15 and 17 showed severe foaming during
reconstitution as compared to the formulation of Example 19. As
seen from Table 9, the stability after the dissolution was similar
for each formulation. But, when sodium chloride was included as the
stabilizer, the stability of the long-acting hGH conjugate was a
little higher.
(5) Analysis of Solubility of Lyophilized Formulation Depending on
Density of Lyophilized Substance and Concentration of Long-Acting
hGH Conjugate
Using the formulation of Example 19 (20 mM sodium citrate, pH 5.2,
5% (w/v) mannitol, 5 mM histidine, 0.005% (w/v) polysorbate 80) of
Test Example 1-(4), the solubility of the lyophilized substance
depending on the long-acting hGH conjugate concentration was
analyzed. Preformulations were prepared with the compositions
described in Table 10 and then lyophilized. During the
lyophilization, the preformulation was diluted 1-fold, 1/2-fold and
1/4-fold using distilled water. The lyophilization consisted of
primary drying and secondary drying steps. The temperature gradient
of the lyophilization is shown in FIG. 1. The reconstitution was
conducted by dissolving the lyophilized formulation with distilled
water of the same volume as that of the formulation before the
lyophilization. The reconstitution was performed using an auto
shaker set to 60.degree. and 30 rpm. The time required for complete
dissolution is given in Table 11.
TABLE-US-00010 TABLE 10 Sugar alcohol and other Conc. Buffer Salt
stabilizer Surfactant Ex. 20 39.0 20 mM sodium -- 5% mannitol
0.005% mg/mL citrate (pH 5.2) 5 mM histidine polysorbate 80 Ex. 21
48.8 20 mM sodium -- 5% mannitol 0.005% mg/mL citrate (pH 5.2) 5 mM
histidine polysorbate 80 Ex. 22 58.5 20 mM sodium -- 5% mannitol
0.005% mg/mL citrate (pH 5.2) 5 mM histidine polysorbate 80
TABLE-US-00011 TABLE 11 Example 20 Example 21 Example 22 Dilution
factor 1 1/2 1/4 1 1/2 1/4 1 1/2 1/4 Dissolution time (sec) 10 10 5
15 10 5 15 10 10
As seen from Table 11, it was confirmed that the dissolution rate
is improved when the density of the lyophilized substance is
decreased through dilution. Also, the dissolution rate increased
similarly when the concentration of the long-acting hGH conjugate
in the formulation (20 mM sodium citrate, 5% mannitol, 5 mM
histidine, 0.005% polysorbate 80) was increased from 39.0 mg/mL to
48.8 mg/mL and to 58.5 mg/mL.
(6) Setting of Temperature Gradient for Lyophilization Process
The temperature gradient in Test Example 1-(1) (FIG. 1) was changed
by increasing the primary drying time from 10 hours to 20 hours and
subdividing the temperature of the primary drying step (4.degree.
C.) into two stages of -20.degree. C. and -5.degree. C. (FIG. 2).
In the former temperature gradient, disruption of the lyophilized
substance occurred because 3-5% of water remained in the
lyophilized substance. When the temperature gradient was changed to
that shown in FIG. 2, complete lyophilization could be achieved
even with a larger volume (.about.5 mL).
(7) Analysis of Solubility of Lyophilized Formulation of
Long-Acting hGH Conjugate Considering Osmotic Pressure
Using the formulation (20 mM sodium acetate, pH 5.6, 5% (w/v)
mannitol, 150 mM sodium chloride, 0.005% (w/v) polysorbate 80) of
Test Example 1-(1) and (2), the concentration of the stabilizer was
set considering osmotic pressure and the solubility of the
lyophilized substance was analyzed. Preformulations were prepared
as described in Table 12 and then lyophilized.
During the lyophilization, the preformulation was diluted 1/2-fold
using distilled water. The lyophilization consisted of primary
drying and secondary drying steps. The temperature gradient of the
lyophilization was set as shown in FIG. 2. The reconstitution was
conducted using distilled water containing 0.9% benzyl alcohol,
which has the same volume as that of the formulation before the
dilution.
The reconstitution was performed using an auto shaker set to
60.degree. and 30 rpm. The time required for complete dissolution
and the osmotic pressure measured after the reconstitution are
shown in Table 13.
TABLE-US-00012 TABLE 12 Sugar alcohol and other Conc. Buffer Salt
stabilizer Surfactant Ex. 23 58.5 mg/mL 20 mM sodium 150 mM 5%
0.005% acetate (pH NaCl mannitol polysorbate 5.6) 80 Ex. 24 58.5
mg/mL 20 mM sodium 150 mM -- 0.005% acetate (pH NaCl polysorbate
5.6) 80 Ex. 25 58.5 mg/mL 20 mM sodium -- 5% 0.005% acetate (pH
mannitol polysorbate 5.6) 80 Ex. 26 58.5 mg/mL 20 mM sodium 75 mM
2.5% 0.005% acetate (pH NaCl mannitol polysorbate 5.6) 80
TABLE-US-00013 TABLE 13 Example 23 Example 24 Example 25 Example 26
Dissolution time 30 180 15 60 (sec) Osmotic pressure 662 338 365
355 (mOsm/Kg)
As seen from Table 13, the dissolution time was increased greatly
when mannitol was removed. The osmotic pressure was higher than the
isotonic range of 280-320 mOsm/Kg when the concentration of
mannitol was 5% (w/v).
(8) Analysis of Solubility and Osmotic Pressure of Lyophilized
Formulation of Long-Acting hGH Conjugate Depending on Mannitol
Concentration
Using the formulation (20 mM sodium acetate, pH 5.6, 5% (w/v)
mannitol, 0.005% (w/v) polysorbate 80) of Test Example 1-(7), the
solubility and osmotic pressure of the lyophilized substance
depending on mannitol concentration were analyzed.
Preformulations were prepared as described in Table 14 and then
lyophilized. The methods and conditions of the lyophilization and
reconstitution were the same as described in Test Example 1-(7).
The time required for complete dissolution and the osmotic pressure
measured after the reconstitution are shown in Table 15.
TABLE-US-00014 TABLE 14 Sugar alcohol and other Conc. Buffer Salt
stabilizer Surfactant Ex. 23 58.5 20 mM sodium 150 mM 5% 0.005%
mg/mL acetate (pH NaCl mannitol polysorbate 5.6) 80 Ex. 25 58.5 20
mM sodium -- 5% 0.005% mg/mL acetate (pH mannitol polysorbate 5.6)
80 Ex. 27 58.5 20 mM sodium -- 4.5% 0.005% mg/mL acetate (pH
mannitol polysorbate 5.6) 80 Ex. 28 58.5 20 mM sodium -- 4% 0.005%
mg/mL acetate (pH mannitol polysorbate 5.6) 80 Ex.29 58.5 20 mM
sodium -- 3.5% 0.005% mg/mL acetate (pH mannitol polysorbate 5.6)
80
TABLE-US-00015 TABLE 15 Ex. 23 Ex. 25 Ex. 27 Ex. 28 Ex. 29
Dissolution time 30 20 22 25 27 (sec) osmotic pressure 632 350 318
291 258 (mOsm/Kg)
As seen from Table 15, isotonic osmotic pressure was observed when
the concentration of mannitol was in the range from 4 to 4.5%.
Although the dissolution time was longer as the mannitol
concentration was lower, the change was smaller as compared to when
sodium chloride was included.
(9) Analysis of Storage Stability of Lyophilized Formulation of
Long-Acting hGH Conjugate at 4.degree. C. and 25.degree. C.
Using the formulation (20 mM sodium acetate, pH 5.6, 4% (w/v)
mannitol, 0.005% (w/v) polysorbate 80) of Test Example 1-(8), the
storage stability of the lyophilized substance was analyzed at
4.degree. C. and 25.degree. C. After storing the lyophilized
formulation at 4.degree. C. and 25.degree. C. for 6 months, the
stability was evaluated by ion exchange chromatography (IE-HPLC)
after reconstitution. The initial solution for reconstitution was
stored at 25.degree. C. for 4 weeks in liquid state and then was
evaluated by ion exchange chromatography (IE-HPLC) after
reconstitution. In Table 16, IE-HPLC (%) indicates the purity of
the long-acting hGH conjugate in the lyophilized substance at the
given time. In Table 17, IE-HPLC (%) indicates the purity of the
long-acting hGH conjugate in the reconstituted liquid
formulation.
TABLE-US-00016 TABLE 16 IE-HPLC (%) Month 0 Month 3 Month 6
4.degree. C. 96.6 96.5 96.2 25.degree. C. 96.5 95.6 96.0
TABLE-US-00017 TABLE 17 IE-HPLC (%) Week 0 Week 1 Week 2 Week 4
96.6 93.1 89.4 87.7
As seen from Table 16, the stability of the lyophilized substance
was maintained even after storing at 4.degree. C. and 25.degree. C.
for 6 months. Also, the stability of the reconstituted formulation
was maintained even after storing at 25.degree. C. for 2 weeks.
Test Example 2
Evaluation of Liquid Formulation of Long-Acting hGH Conjugate
(1) Analysis of Stability of Liquid Formulation of Long-Acting hGH
Conjugate Depending on pH, Buffer, Isotonic Agent and Sugar Alcohol
Concentration
The effect of a buffer, an isotonic agent and the sugar alcohol
concentration on the stability of the long-acting hGH conjugate was
tested. Formulations prepared as described in Table 18 were stored
at 25.degree. C. for 0-4 weeks and then analyzed by ion exchange
chromatography and size exclusion chromatography. In Tables 19 and
20, IE-HPLC (%) and SE-HPLC (%) indicate the residual rate of the
long-acting hGH conjugate relative to the initial value,
respectively (area %/start area %).
TABLE-US-00018 TABLE 18 Sugar Long-acting Iso- alcohol hGH tonic
and other conjugate pH Buffer agent stabilizer Surfactant Ex. 30
58.5 mg/mL 5.2 20 mM 75 mM 2% 0.005% sodium NaCl mannitol
polysorbate acetate 80 Ex. 31 58.5 mg/mL 5.2 20 mM -- 4% 0.005%
sodium mannitol polysorbate acetate 80 Ex. 32 58.5 mg/mL 5.6 20 mM
75 mM 2% 0.005% sodium NaCl mannitol polysorbate acetate 80 Ex. 33
58.5 mg/mL 5.6 20 mM -- 4% 0.005% sodium mannitol polysorbate
acetate 80 Ex. 34 58.5 mg/mL 5.6 20 mM 75 mM 2% 0.005% histi- NaCl
mannitol polysorbate dine 80 Ex. 35 58.5 mg/mL 5.6 20 mM -- 4%
0.005% histi- mannitol polysorbate dine 80
TABLE-US-00019 TABLE 19 IE-HPLC (%) Week 0 Week 1 Week 2 Week 3
Week 4 Example 30 100.0 98.5 97.0 95.3 91.9 Example 31 100.0 98.9
96.9 94.8 91.9 Example 32 100.0 96.9 94.9 92.2 87.2 Example 33
100.0 97.8 96.7 94.7 90.2 Example 34 100.0 97.8 94.7 92.4 86.5
Example 35 100.0 96.1 93.0 88.4 82.9
TABLE-US-00020 TABLE 20 SE-HPLC (%) Week 0 Week 1 Week 2 Week 3
Week 4 Example 30 100.0 99.6 99.4 99.3 99.1 Example 31 100.0 100.2
100.0 99.9 99.8 Example 32 100.0 99.6 99.6 99.5 99.4 Example 33
100.0 99.6 99.6 99.4 99.3 Example 34 100.0 100.1 100.1 99.9 99.7
Example 35 100.0 99.9 99.8 99.7 99.3
The IE-HPLC result showed that the long-acting hGH conjugate shows
good stability under the condition of 20 mM sodium acetate (pH
5.2). And, the SE-HPLC result showed that the long-acting hGH
conjugate was the most stable under the condition of 20 mM sodium
acetate (pH 5.2) and 4% (w/v) mannitol.
(2) Analysis of Stability of Liquid Formulation of a Long-Acting
hGH Conjugate Depending on Buffer
The effect of a buffer as a stabilizer on the stability of the
long-acting hGH conjugate was tested. Using the formulation (pH
5.2, 4% mannitol, 0.005% polysorbate 80) of Test Example 2-(1),
formulations were prepared as described in Table 21. After storing
at 25.degree. C. for 0-4 weeks, the stability was analyzed by
IE-HPLC and SE-HPLC. In Tables 22 and 23, IE-HPLC (%) and SE-HPLC
(%) indicate the residual rate of the long-acting hGH conjugate
relative to the initial value, respectively (area %/start area
%).
TABLE-US-00021 TABLE 21 Sugar Long-acting Iso- alcohol hGH tonic
and other conjugate pH Buffer agent stabilizer Surfactant Ex. 36
10.0 mg/mL 5.2 20 mM -- 4% mannitol 0.005% sodium polysorbate
citrate 80 Ex. 37 10.0 mg/mL 5.2 20 mM -- 4% mannitol 0.005% sodium
polysorbate acetate 80 Ex. 38 10.0 mg/mL 5.2 20 mM -- 4% mannitol
0.005% histi- polysorbate dine 80 Ex. 39 58.5 mg/mL 5.2 20 mM -- 4%
mannitol 0.005% sodium polysorbate citrate 80 Ex. 31 58.5 mg/mL 5.2
20 mM -- 4% mannitol 0.005% sodium polysorbate acetate 80 Ex. 40
58.5 mg/mL 5.2 20 mM -- 4% mannitol 0.005% histi- polysorbate dine
80
TABLE-US-00022 TABLE 22 IE-HPLC (%) Week 0 Week 1 Week 2 Week 3
Week 4 Example 36 100.0 97.4 96.1 94.9 93.1 Example 37 100.0 98.3
96.5 92.7 88.5 Example 38 100.0 97.9 96.4 93.9 91.7 Example 39
100.0 98.4 96.7 94.8 92.7 Example 31 100.0 97.9 96.3 94.6 92.8
Example 40 100.0 98.2 96.3 94.2 92.2
TABLE-US-00023 TABLE 23 SE-HPLC (%) Week 0 Week 1 Week 2 Week 3
Week 4 Example 36 100.0 98.9 98.7 98.7 98.4 Example 37 100.0 98.6
98.6 98.8 98.1 Example 38 100.0 98.7 98.8 98.8 98.6 Example 39
100.0 99.4 99.2 99.2 98.9 Example 31 100.0 99.8 99.1 99.2 98.8
Example 40 100.0 100.0 99.2 99.4 99.1
The IE-HPLC result showed that 10.0 mg/mL long-acting hGH conjugate
was the most stable under the condition of sodium citrate. And, the
SE-HPLC result showed that 10.0 mg/mL long-acting hGH conjugate
showed good stability in the order of histidine and sodium
citrate.
The IE-HPLC result showed that 58.5 mg/mL long-acting hGH conjugate
showed good stability in the order of sodium acetate and sodium
citrate. And, the SE-HPLC result showed that 58.5 mg/mL long-acting
hGH conjugate showed good stability in the order of histidine and
sodium citrate.
When the concentration of the long-acting hGH conjugate was in the
range from 10 mg/mL to 58.5 mg/mL, the best stability was observed
under the condition of 20 mM sodium citrate (pH 5.2), 4% mannitol
and 0.005% polysorbate 80.
It will be apparent to those skilled in the art that various
modifications and changes may be made without departing from the
scope and spirit of the invention. Therefore, it should be
understood that the above embodiment is not limitative, but
illustrative in all aspects. The scope of the invention is defined
by the appended claims rather than by the description preceding
them, and therefore all changes and modifications that fall within
metes and bounds of the claims, or equivalents of such metes and
bounds are therefore intended to be embraced by the claims.
* * * * *